Many viral proteins are involved in the regulation of viral genome replication or gene transcription, and some proteins play a role in both processes To facilitate the development of effective therapies based on such proteins, it is necessary to distinguish between effects on gene transcription and replication. Current systems employed for such assays evaluate effects on replication using hybridization or amplification techniques for directly assessing the level of plasmid in a cell sample. Such techniques involve plasmid isolation and digestion, and are too time-consuming and costly for most routine screening purposes. Other techniques employ separate plasmids, each containing a reporter gene, but such systems do not reflect the natural environment and are limited in the ability to assess the two processes, since replication of the two plasmids is fully independent.
One pathogen that employs proteins that modulate both transcription and replication is the papillomavirus. Human papillomaviruses (HPVs) infect squamous epithelial cells, and can induce a variety of benign papillomas or neoplasias. Certain HPV types are commonly associated with benign genital warts, which rarely progress to cancer. However, other HPV types are associated with intraepithelial neoplastic lesions that can progress to malignancies. There is currently no adequate treatment or preventive therapy for such conditions.
The life cycle of HPV can generally be divided into three phases. In the first phase, HPV infects basal cells and the viral genome is established as a stable, low-copy episome. In the second phase, the viral genome is maintained at 50-100 copies per cell, and replicates with the cellular DNA. During the third phase the infected cells undergo differentiation and the viral genome is amplified to several thousand copies per cell. Viral late gene expression is also induced at this time.
To facilitate the development of improved therapies for HPV-associated conditions, it is necessary to gain a further understanding of the molecular mechanisms that regulate events in each phase of the HPV life cycle. Studies to date have identified two proteins, E1 and E2, which are required for viral replication and gene transcription (see Ustav and Stenlund, EMBO J. 10:449-457, 1991). E1 is a 73 kD protein that binds ATP and the HPV origin of replication, and exhibits helicase and origin unwinding activities (see, e.g., Blitz and Laimins, J. Virol. 65:649-656, 1991; Lambert, J. Virol. 65:3417-3420, 1991). E2 is a 42 kD transcriptional activator that binds to the sequence (ACCN.sub.6 GGT; SEQ ID NO: 1) and also forms a complex with E1. However, as a result of this complex formation, and because these proteins appear to have multiple functions, the specific roles of each protein in the HPV life cycle remain unclear. Further work needs to be performed to understand the roles of E1. E2 and other proteins in the regulation of HPV transcription and replication and to identify agents that modulate the activity of such proteins. Such studies have been hampered by the lack of an in vitro system for the separate evaluation of both processes.
Accordingly, there is a need in the art for a system that permits independent assay of transcription and replication, particularly for viral systems. The present invention fulfills this need and further provides other related advantages.